Peptide antigens presented by class I MHC molecules and recognized by CD8 T lymphocytes on human melanoma cells have been defined and are the subject of a number of clinical trials to develop immunotherapeutic vaccines. However, no consensus methodology exists for delivery of peptide antigens to stimulate the development of effective CTL. In addition, many of these antigens are also expressed in normal melanocytes, and it is not clear how self-tolerance compromises the intrinsic anti-tumor immune response and the vaccination induced response. Because comprehensive evaluation of these issues in clinical trials is difficult, we developed a preclinical model using transgenic mice expressing a human class I MHC molecule and a peptide antigen derived from murine tyrosinase (Tyr369) that is very homologous to its human counterpart. In the previous funding period, we established key elements in this model system, and used it to begin to assess the effectiveness of MDP specific immune responses against tumor and their modification by self-tolerance. In addition, we developed peptide pulsed DC as an immunization strategy that enables evaluation of issues related to Ag density and route of administration. The latter results have implications for vaccination induced anti-tumor immunity, and also point towards the use of peptide pulsed dendritic cells as a model to provide basic insights into the stimulation of CD8 T cell responses. Finally, we also developed two strains of transgenic mice that express TCR for Tyr369, but differ in their structural avidity. These new strains offer the possibility to substantially increase understanding in 3 areas: mechanisms of self-tolerance to MDP, vaccination induced immunity, and immunity to Ags expressed on tumors. In the next funding period, we propose a more extensive evaluation of several of these issues. Accordingly, the specific aims of this proposal are: 1) To determine how self-tolerance to the melanocyte differentiation protein tyrosinase influences the development and activity of tyrosinase specific CD8 T cells; 2) To analyze the size and avidity of CD8+ T cell responses to Tyr369-pulsed CD40L-activated DC; 3) 3.Toanalyze the basis for regional immunity elicited with Tyr369-pulsed CD40L-activated DC, and; 4) To examine immunity to Tyr369 elicited by and directed against a tyrosinase+ tumor. This work will lead to important insights into the clinical use of MDP Ags and DC for tumor immunotherapy.